System for pH-neutral stable electrophoresis gel

ABSTRACT

A gel and buffer system for gel electrophoresis wherein separation occurs at neutral pH.

This application is a continuation of copending application Ser. No.09/228,875, now U.S. Pat. No. ______, which is a continuation-in-part ofapplication Ser. No. 08/730,678, now U.S. Pat. No. 5,922,185, which is acontinuation-in-part of application Ser. No. 08/221,939, filed Mar. 31,1994, now U.S. Pat. No. 5,578,180.

This invention relates to techniques for gel electrophoresis. Moreparticularly this invention relates to a novel system for gelelectrophoresis at approximately neutral pH.

BACKGROUND OF THE INVENTION

Gel electrophoresis is a common procedure for the separation ofbiological molecules, such as deoxyribonucleic acid (DNA), ribonucleicacid (RNA), polypeptides and proteins. In gel electrophoresis, themolecules are separated into bands according to the rate at which animposed electric field causes them to migrate through a filtering gel.

The basic apparatus used in this technique consists of a gel enclosed ina glass tube or sandwiched as a slab between glass or plastic plates.The gel has an open molecular network structure, defining pores whichare saturated with an electrically conductive buffered solution of asalt. These pores through the gel are large enough, to admit passage ofthe migrating macromolecules.

The gel is placed in a chamber in contact with buffer solutions whichmake electrical contact between the gel and the cathode or anode of anelectrical power supply. A sample containing the macromolecules and atracking dye is placed on top of the gel. An electric potential isapplied to the gel causing the sample macromolecules and tracking dye tomigrate toward the bottom of the gel. The electrophoresis is halted justbefore the tracking dye reaches the end of the gel. The locations of thebands of separated macromolecules are then determined. By comparing thedistance moved by particular bands in comparison to the tracking dye andmacromolecules of known mobility, the mobility of other macromoleculescan be determined. The size of the macromolecule can then be calculated.

The rate of migration of macromolecules through the gel depends uponthree principle factors: the porosity of the gel; the size and shape ofthe macromolecule; and the charge density of the macromolecule. It iscritical to an effective electrophoresis system that these three factorsbe precisely controlled and reproducible from gel to gel and from sampleto sample. However, maintaining uniformity between gels is difficultbecause each of these factors is sensitive to many variables in thechemistry of the gel system.

Polyacrylamide gels are commonly used for electrophoresis.Polyacrylamide gel electrophoresis or PAGE is popular because the gelsare optically transparent, electrically neutral and can be made with arange of pore sizes. The porosity of a polyacrylamide gel is in partdefined by the total percentage of acrylamide monomer plus crosslinkermonomer (“% T”) it contains. The greater the concentration, the lessspace there is between strands of the polyacrylamide matrix and hencethe smaller the pores through the gel. An 8% polyacrylamide gel haslarger pores than a 12% polyacrylamide gel. An 8% polyacrylamide gelconsequently permits faster migration of macromolecules with a givenshape, size and charge density. When smaller macromolecules are to beseparated, it is generally preferable to use a gel with a smaller poresize such as a 20% gel. Conversely for separation of largermacromolecules, a gel with a larger pore size is often used, such as an8% gel.

Pore size is also dependent upon the amount of crosslinker used topolymerize the gel. At any given total monomer concentration, theminimum pore size for a polyacrylamide gel is obtained when the ratio oftotal monomer to crosslinker is about 20:1, (the usual expression forthis ratio would be “5% C”).

Several factors may cause undesirable variation in the pore size ofgels. Pore size can be increased by incomplete gel polymerization duringmanufacture. Hydrolysis of the polyacrylamide after polymerization cancreate fixed negative charges and break down the crosslinks in the gel,which will degrade the separation and increase the pore size. An idealgel system should have a reproducible pore size and no fixed charge (orat least a constant amount) and should be resistant to change inchemical characteristics or the pore size due to hydrolysis.

The size of the macromolecule varies between different macromolecules;the smaller and more compact the macromolecule the easier it will be forthe macromolecule to move through the pores of a given gel. Given aconstant charge density, the rate of migration of a macromolecule isinversely proportional to the logarithm of its size.

For accurate and reproducible electrophoresis, a given type ofmacromolecule should preferably take on a single form in the gel. Onedifficulty with maintaining uniformity of the shape of proteins duringgel electrophoresis is that disulfide bonds can be formed by oxidationof pairs of cysteine amino acids. Different oxidized forms of theprotein then have different shapes and, therefore, migrate through thegel run with slightly different mobilities (usually faster than acompletely reduced protein, since the maximum stokes radius and minimummobility should occur with a completely unfolded form). A heterogeneousmixture of forms leads to apparent band broadening. In order to preventthe formation of disulfide bonds, a reducing agent such asdithiothreitol (DTT) is usually added to the samples to be run. Theshape of DNA and RNA macromolecules is dependent on temperature. Inorder to permit electrophoresis on temperature-dependent DNA and RNAmolecules in their desired form, separations are done at a controlledtemperature.

The charge density of the migrating molecule is the third factoraffecting its rate of migration through the gel—the higher the chargedensity, the more force will be imposed by the electric field upon themacromolecule and the faster the migration rate subject to the limits ofsize and shape. In SDS PAGE electrophoresis, the charge density of themacromolecules is controlled by adding sodium dodecyl sulfate (SDS) tothe system. SDS molecules associate with the macromolecules and impart auniform charge density to them, substantially negating the effects ofany innate molecular charge. Unlike proteins, the native charge densityof DNA and RNA is generally constant, due to the uniform occurrence ofphosphate groups. Thus, charge density is not a significant problem inelectrophoresis of DNA and RNA.

SDS PAGE gels are usually poured and run at basic pH. The most commonPAGE buffer system employed for the separation of proteins is thatdeveloped by Ornstein (1) and modified for use with SDS by Laemmli (2).Laemmli, U.K. (1970) Nature 227, 680-686. The Laemmli buffer systemconsists of 0.375 M tris (hydroxy methyl) amino-methane (Tris), titratedto pH 8.8. with HCl, in the separating gel. The stacking gel consists of0.125 M Tris, titrated to pH 6.8. The anode and cathode running bufferscontain 0.024 M Tris, 0.192 M glycine, 0.1% SDS. An alternative buffersystem is disclosed by Schaegger and von Jagow. Schaegger, H. and vonJagow, G., Anal. Biochem. 1987, 166, 368-379. The stacking gel contains0.75 M Tris, titrated to pH 8.45 with HCl. The separating gel contains0.9 M Tris, titrated to pH 8.45 with HCl. The cathode buffer contains0.1 M Tris, 0.1M N-tris(hydroxymethyl)methylglycine (tricine), 0.1% SDS.The anode buffer contains 0.2 M Tris, titrated to pH 8.9 with HCl. Forboth of these systems Tris is the “common ion” which is present in thegel and in the anode and cathode buffers.

In the Laemmli system, the pH of the trailing phase in the stacking gelis about 8.9. In the separating gel, the trailing phase pH is about 9.7.At this pH, primary amino groups of proteins react readily withunpolymerized acrylamide, thiol groups are more subject to oxidation todisulfides, or reaction with unpolymerized polyacrylamide, than atneutral pH and acrylamide itself is subject to hydrolysis.

The shape of the DNA and RNA macromolecules is also dependent on afourth important factor, temperature. The temperature-dependent shape ofDNA and RNA is caused by the interaction of two macromoleculescontaining complementary sequences and the interaction of complementarysequences in a single macromolecule. Some techniques require that theDNA remain in its double-stranded form. Typically, such separations aredone in Tris borate ethylene diamine tetra-acetic acid (TBE) buffer,consisting of 0.09 M Tris, 0.09 M boric acid, and 0.002 M ethylenediamine tetra-acetic acid (EDTA) on either polyacrylamide or agarosegels. In general, these separations are done at lower temperatures tomaintain the double-stranded structure. In the absence of denaturants,DNA's and RNA's structure is fairly stable and not significantlyaffected by temperature.

In other techniques, dissociation of the two DNA strands (known as“melting”) is utilized to effect the separation. Such methods requirecareful temperature control in order to produce a consistent separation.One method, non-isotopic single-strand conformational polymorphism(“Cold SSCP”), utilizes a dissociative sample buffer with heat to meltthe strands, a TBE buffer, and a polyacrylamide gel. In Cold SSCP,temperatures of 4 to 35° C. are used to allow variable-conformationrenaturation to occur between mutant strands, and temperature changes ofonly a few degrees can significantly alter the number of mutants seen.See Hongyo, et al., Nucleic Acids Research, 21, 3637 (1993). Anothermethod, employed in DNA sequence analysis, typically utilizes TBEbuffers containing 6 to 8 M urea and/or 2 to 12 M formamide, andelevated temperatures. It is important that the temperature remain highenough—typically 45 to 55° C.—to maintain fully melted DNA or RNA. Gelsare usually polyacrylamide and sometimes substituted acrylamidepolymers. For example, certain alkyl-substituted polyacrylamide gels aredescribed in Shoor et al., U.S. Pat. No. 5,055,517.

These DNA and RNA separation methods are characterized by the use ofcontinuous buffer systems, which use the same buffer species andgenerally, but not necessarily, in the same concentrations in the gel,the anode chamber, and the cathode chamber. These buffers usually arecomprised of Tris and boric acid with EDTA added to inhibit hydrolyticenzyme activity. The TBE buffer system typically does not provide goodstability when used in pre-cast gels, made and stored for periods ofweeks at 4° C. The polymer tends to break down, generating a fixedcharge which leads to distortion particularly at the cathode end of thegel where resolution is especially important. Urea also tends to breakdown under alkaline pH at 4° C. When large concentrations of urea arepresent, the ionic breakdown products can be present at a large enoughconcentration to disrupt the separation and cause loss of resolution.

Other buffer systems for DNA and RNA separations employ Tris/acetate,Tris/phosphate, and Tris/glycylglycine. While these buffer systems maybe formulated near pH 7, the pK_(a) of Tris causes them to shift to analkaline pH during electrophoresis especially near the cathode. Theapplicants have found that the polyacrylamide and urea tend to breakdown during electrophoresis for DNA sequencing due to the hightemperatures (50° C.) employed for several hour runs when Tris is usedas the buffering base. This breakdown leads to higher current and lowerresolution than might be obtained with a neutral pH buffer system, sothat the DNA sequence read length is reduced and read errors areincreased.

The need for uniformity and predictability is magnified in precastelectrophoresis gels which are manufactured by an outside vendor andthen shipped to the laboratory where the electrophoresis will beperformed. Precast gels must control the properties discussed above andthey must be able to maintain this control throughout shipping andstorage. The shelf life of many precast gels is limited by the potentialfor hydrolysis of acrylamide and/or buffer constitution during storageat the high pH of the gel buffer.

It is a disadvantage of a high pH gel that the polyacrylamide gel issubject to degradation by hydrolysis and has a limited shelf-life.

It is a further disadvantage of a high pH gel that proteins reactreadily with unpolymerized acrylamide which may interfere withsubsequent analysis of the protein such as peptide sequencing.

It is a still further disadvantage of a high pH gel that thiol groupsare subject to oxidation to disulfides causing a decreased resolution ofseparated macromolecules.

It is a further disadvantage of a high pH gel that buffer constituentssuch as urea break down readily.

SUMMARY OF THE INVENTION

It is an object of this invention to produce a neutral gel system thatreduces protein reaction with unpolymerized acrylamide thereby enhancingyield and resolution.

It is a further object of this invention to produce a neutral gel systemthat prevents formation of disulfides from free thiol groups therebyenhancing yield and resolution.

It is also an object of this invention to produce a neutral gel systemthat reduces degradation of the polyacrylamide gel by hydrolysis therebyincreasing the stability of a gel during electrophoresis and the usefulshelf-life of a precast gel, and better resolution.

It is also an object of this invention to produce a neutral gel systemthat reduces breakdown of buffer constituents, such as urea.

In accordance with this invention, applicants describe a gel and buffersystem wherein separation occurs at neutral pH and proteins remaincompletely reduced. Applicants also describe a gel and buffer systemwherein storage of the gel and subsequent electrophoresis ofmacromolecules (such as DNA, RNA, polypeptides and proteins) occurs atneutral pH. The above and other objects and advantages of the presentinvention will be apparent upon consideration of the following detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

Applicants describe a gel and buffer system wherein separation occurs atneutral pH and proteins remain completely reduced. Advantageously, atthis neutral pH, primary amino groups of proteins react less readilywith unpolymerized acrylamide because protonation of protein aminogroups greatly reduces their reactivity to acrylamide or other relatedblocking agents. Furthermore, at this neutral pH, thiol groups are lesssubject to oxidation than at higher pH and polyacrylamide itself is lesssubject to hydrolysis.

The result is a gel system with improved stability of the gel matrix andstock solutions. Gels prepared according to this system can be storedunder refrigeration for over a year without loss of performance due toacrylamide hydrolysis. Also, stock buffers without reducing agents andstock gel solutions without polymerization initiator can be stored forat least several weeks at room temperature with no loss of performance.An additional benefit is that a single gel recipe, using the same bufferfor the stacking and separating gels, can be used with two differentrunning buffers to give two separation systems. Using this feature, an8% gel, for example, can cover a protein separation range of 2 to 200kDa.

In one embodiment of this invention a polyacrylamide gel of betweenabout 3% and about 25% (% T) acrylamide is polymerized using from about1% to about 6% crosslinker (% C) using a gel buffer comprising a primaryorganic amine or substituted amine with a PK_(a) near neutrality,titrated with approximately half as much HCl (on a molar basis), so thatthe pH of the buffer is approximately neutral. In a preferred embodimentthe gel is polymerized using from about 2% to about 5% crosslinker (% C)using a gel buffer comprising bis-(2-hydroxyethyl) iminotris(hydroxymethyl) methane (Bis-Tris) titrated with HCl. Differentseparation characteristics can be obtained by running the gel witheither a 3-(N-morpholino) propanesulfonic acid (MOPS) or2-(N-morpholino) ethanesulfonic acid (MES), buffer. 2 mM to 10 mMthioglycolic acid (TGA) or 2 mM to 10 mM sodium bisulfite is added tothe running buffer to maintain a reducing environment in the gel duringelectrophoresis.

Applicants also describe another gel and buffer system for separation ofmacromolecules (including DNA, RNA, polypeptides and proteins) whereinseparation occurs at neutral pH. This gel and buffer system may be adiscontinuous or continuous buffer system, but is particularly useful ina continuous system. A continuous buffer system is one using the samebuffer species and generally, but not necessarily, in the sameconcentrations in the gel, the anode chamber and the cathode chamber.This gel and buffer system permits higher resolution duringelectrophoresis when alkaline-labile compounds such as polyacrylamideand urea are present. This gel and buffer system also permits higherresolution when elevated temperatures are used. Advantageously, at thisneutral pH, urea is less subject to hydrolysis. Furthermore,polyacrylamide itself is less subject to hydrolysis.

This gel and buffer system also possesses improved stability of the gelmatrix and stock solutions. Gels prepared according to this system canbe stored under refrigeration for over a year without loss ofperformance due to acrylamide hydrolysis. Also, stock buffers and stockgel solutions without polymerization initiator can be stored for atleast several weeks at room temperature with no loss of performance.

In an embodiment of this gel and buffer system an electrophoresis gel isuniformly saturated with a gel buffer solution comprising a primaryorganic amine or substituted amine with a pK_(a) near neutrality,titrated with approximately an equimolar amount of acid or zwitterioniccompound, so that the pH of the buffer is between about pH 6 and pH 8,preferably between about pH 6.5 to pH 7.5, and most preferably 6.5 to7.0. The electrophoresis gel may be any agarose or polyacrylamide gel.Preferably, the electrophoresis gel comprises between 3% and 25% (% T)acrylamide polymerized using from about 1% to about 6% cross linker (%C). More preferably, this polyacrylamide gel is polymerized using fromabout 2% to about 5% crosslinker (% C). Preferably, the amine comprisesBis-Tris or N-(2-hydroxyethyl) morpholine, and most preferably,Bis-Tris. Suitable acids and zwitterionic compounds are hydrochloricacid, tricine, acetic acid, piperazine-N,N′-2-ethanesulfonic acid,3-(N-morpholino)-propanesulfonic acid, 2-(N-morpholino)-ethanesulfonicacid, N-(2-acetamido)-2-aminoethanesulfonic acid,2-(N-morpholino)-2-hydroxypropanesulfonic acid,N-tris-(hydroxymethyl)-2-ethanesulfonic acid,N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid,N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid, and3-(N-tris-(hydroxymethyl) methylamino)-2-hydroxypropanesulfonic acid.Tricine, 2-(N-morpholino)-ethanesulfonic acid, andpiperazine-N,N′-2-ethanesulfonic acid are preferred for use in thebuffer for a continuous gel and buffer system for separation of DNA andRNA because the resulting system has separation characteristics similarto the commonly used TBE gel systems. Tricine is most preferred for thatuse. Preferably, the gel buffer comprises Bis-Tris titrated withtricine.

In a gel and buffer system, current increases and migration ratesdecline as the performance of the gels decline. The increase in currenthas been attributed to alkaline-catalyzed hydrolysis of urea present at36% to 42% concentration. Any breakdown in a neutral substance presentat a large concentration, which produces a charged species will tend todisrupt the electrophoresis. This disruption arises from the extracurrent produced, which in turn increases joule heating without aidingthe separation. In addition, a discontinuity arises from the anionic andcationic hydrolysis products forming in the gel that are not present inthe cathode and anode buffers. Hydrolysis of gel buffer species oradditives takes place independently from the gel matrix composition. Thedecrease in migration rate may be attributed to higher fixed charge inthe gel caused by alkaline-catalyzed hydrolysis of the gel'spolyacrylamide. The fixed charge leads to significant counter-flow ofwater, which can retard a macromolecule's-migration rate. It has beenfound that problems of gel instability producing lower resolution,increased current, decreased migration rates can be solved with gelsbuffered near neutrality and with buffer substances having a pK_(a) nearneutrality. Such buffering systems improve the performance of fresh orpre-cast polyacrylamide gels, and fresh or pre-cast gels containingalkaline-labile materials, such as urea or formamide, even when the gelsare made with base-stable polymers.

The inventors also have discovered the value of using different buffersubstances in the cathode, gel, and anode buffers. A group ofsubstitutions relating to cost and throughput have been discovered. Theanionic substance used in the gel or the cathode buffer need not bepresent in the anode buffer since the anions do not migrate out of theanode buffer. In fact, the use of chloride or other strong acids in theanode buffer serves to increase the conductance of the buffer, therebyincreasing the net voltage drop across the gel and decreasing run times.Such acids are also typically much less expensive as compared to thoseemployed in the cathode buffer. Similarly, the base used to adjust thepH of the cathode buffer need not be the same as that used in the gel.At a neutral pH, sodium hydroxide, Tris, and other organic bases with abasic pK_(a) have a higher conductance and often lower cost thanBis-Tris or other bases with a pK_(a) near neutrality. Using sodium orTris salts in the cathode buffer will also decrease the gel run times.Often, the anode and cathode buffers are used at a higher concentrationthan in the gel, further increasing their conductance and decreasing gelrun times. Thus, using different, more conductive anode and cathodebuffer species than in the gel buffer increases throughput and decreasescosts.

It was also found that Tris or Bis-Tris may be used in the anode bufferwith no visible effect on the separation quality. Because of its higherpK_(a), Tris gradually infiltrates the anode end of the gel increasingthat region's conductance, causing the voltage drop to fall locally.Thus, the macromolecules near the anode slow down and the separationcompresses, while the macromolecules near the cathode experience ahigher voltage drop increasing their migration and relative separation.This effect of Tris actually improves the resolution of macromoleculesat the cathode end of the gel where it is most needed. Tris is thepreferred choice for routine use, because it is available atsignificantly lower cost than Bis-Tris and can improve read lengths.

The preferred embodiment of this invention uses Tris chloride in theanode buffer, the sodium or Tris salt of the acid or zwitterioniccompound in the cathode buffer, and Bis-Tris as the gel buffer amine.For protein and polypeptide separations, the most preferred cathodebuffers are sodium or Tris salts of MOPS and MES, combined with aBis-Tris chloride gel buffer. For DNA and RNA separations, the mostpreferred cathode buffers are sodium or Tris salts of tricine, combinedwith a Bis-Tris tricine EDTA running buffer. When the buffer chambersare small, the most preferred molar concentrations of the cathode andanode buffers are five times that present in the gel buffer. Thesebuffer systems provide the benefits of a neutral pH gel during bothstorage and running, the least cost, and the fastest run times.

These and other embodiments can be understood by reference to thefollowing illustrative and comparative examples.

EXAMPLES

Tris, Bis-Tris, MES, tricine, MOPS and piperazine-N,N′-2-ethanesulfonicacid (PIPES) were purchased from Sigma (St. Louis, Mo.) or ResearchOrganics (Cleveland, Ohio). Thioglycolic acid (TGA), dithiothreitol(DTT) and beta-mercaptoethanol (BME) were from Sigma. All otherchemicals were reagent, “ultra pure” or “electrophoresis grade” fromstandard sources.

In Example 1 through 6, gels were cast in 1 mm thickness mini-gelcassettes from NOVEX (San Diego Calif.) and run in an X-Cell minicell.The Bis-Tris separating gel and stacking gels were prepared from a 30%T/2.5% C acrylamide/BIS stock solution and a 7× Bis-Tris stock solution(2.5M Bis-Tris, 1.5M HCl, pH 6.5). To prepare the separating gel, thestock solutions were blended with ultra pure water to a finalconcentration 8% T, 0.357M Bis-Tris, to which was added 0.2 ul/mlN,N,N′,N′-tetra-methyl-ethylene-diamine (TEMED). After degassing, 2.0ul/ml of a 10% solution of ammonium persulfate (APS) was added, the gelwas immediately poured into the cassette then overlaid with water.Polymerization was allowed to proceed for at least 30 minutes at roomtemperature (RT), the water was removed and a 4% stacking gel applied.The stacking gel was prepared in the same fashion as the separating gel,except that the final concentration obtained was 4% T, the TEMEDconcentration was increased to 0.4 ul/ml and the APS solution increasedto 5.0 ul/ml. MOPS running buffer consisted of 50 mM MOPS, 50 mMBis-Tris (or Tris), 0.1% SDS, 1 mM EDTA. MES running buffer consisted of50 mM MES, 50 mM Bis-Tris (or Tris), 0.1% SDS, 1 mM EDTA. Sample buffer(2×) consisted of 0.25 M Bis-Tris, 0.15 M HCl, 10% (w/v) Glycerol, 2%SDS, 1 mM EDTA, 0.03% Serva Blue G, and 200 mM DTT. Samples containing aset of protein standards were heated for 15 min at 70 degrees beforeapplication. Bovine serum albumin (BSA), chicken egg ovalbumin,alkylated insulin A and B chain, soybean trypsin inhibitor, and bovineerythrocyte carbonic anhydrase were included in the standard. Samplevolume was 5 ul in all cases.

Example 1

The protein standards were separated on an 8% Bis-Tris/Cl gel with MOPSrunning buffer in the absence of a reducing agent. The resultingseparation pattern was very similar to that obtained on an 8%Tris/glycine gel (Laemmli), with proteins 20,000 and smaller remainingin the stack along with the tracking dye. The BSA band was somewhatdiffuse and shifted toward the anode. The Ovalbumin band was alsosomewhat diffuse.

Example 2

The protein standards were separated on an 8% Bis-Tris/Cl gel with MOPSrunning buffer in the presence of TGA in the cathode buffer. Again, theseparation pattern was very similar to that obtained on an 8%Tris/glycine (Laemnli) gel, with proteins 20,000 and smaller remainingin the stack along with the tracking dye. The presence of the reducingagent, 5 mM TGA, in the cathode buffer provided for better resolution ofthe proteins BSA and Ovalbumin compared to the gel run without TGA.

Example 3

The protein standards were separated on an 8% Bis-Tris/Cl gel with MOPSrunning buffer in the presence of sodium bisulfite in the cathodebuffer. Again, the separation pattern was very similar to that obtainedon an 8% Tris/glycine (Laemmli) gel, with proteins 20,000 and smallerremaining in the stack along with the tracking dye. The presence of thereducing agent, 5 mM sodium bisulfite, in the cathode buffer providedfor better resolution of the proteins BSA and Ovalbumin compared to thegel run without sodium bisulfite.

Example 4

The protein standards were separated on an 8% Bis-Tris/Cl gel with MESrunning buffer in the absence of a reducing agent. The proteinseparation was very similar to that obtained from an 12% Tris/tricine(Schaegger) gel. All proteins were resolved from the stack includinginsulin A and B chain (3500 and 2500 daltons, respectively). When thegel is run without TGA, soybean trypsin inhibitor had a more prominentdoublet.

Example 5

The protein standards were separated on an 8% Bis-Tris/Cl gel withBis-Tris/MES running buffer in the presence of TGA in the cathodebuffer. Again, all proteins were resolved from the stack includinginsulin A and B chain (3500 and 2500 daltons, respectively). Thepresence of the reducing agent, 5 mM TGA, in the cathode buffer providedfor better resolution of the protein soybean trypsin inhibitor. Carbonicanhydrase ran as a tight, sharp band under all conditions tested.

Example 6

The protein standards were separated on an 8% Bis-Tris/Cl gel withBis-Tris/MES running buffer in the presence of sodium bisulfite in thecathode buffer. Again, all proteins were resolved from the stackincluding insulin A and B chain (3500 and 2500 daltons, respectively).The presence of the reducing agent, 5 mM sodium bisulfite, in thecathode buffer provided for better resolution of the protein soybeantrypsin inhibitor. Carbonic anhydrase ran as a tight, sharp band underall conditions tested.

Although MES and MOPS were selected as desirable running buffers forprotein separation because the resulting system has separationcharacteristics similar to the commonly used Laemmli and Schaegger gelsystems, it was found that a range of buffers are suitable for use inthis system. Among the additional buffers giving good results were[N-(2-acetamido)]-2-aminoethanesulfonic acid (ACES),2-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPSO),N-Tris-(hydroxymethyl)-2-ethanesulfonic acid (TES),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES), and3-(N-Tris-(hydroxymethyl) methylamino)-2-hydroxypropanesulfonic acid(TAPSO).

All the proteins that exhibit some band broadening and/or mobilityshifts when run in the absence of TGA or sodium bisulfite, have incommon a composition that includes multiple cysteines (BSA, forinstance, has 35 cysteines). On the other hand carbonic anhydrase, whichalways runs cleanly, has no cysteines. Moreover, if the reduced proteinsare alkylated before running, they run as sharp homogeneous bands evenin the absence of a reducing agent.

Cysteine-containing proteins appear to give generally sharper bands inthe Laemmli system than the neutral system, when both are run with 100mm mercaptoethanol or DTT in the sample buffer but without TGA in therunning buffer. Since thiol oxidation is more favored as the pHincreases, it would be expected that the higher pH of the Laemmli systemwould cause oxidation of disulfide to be at least as pronounced as it isin the neutral pH system. However, DTT and similar “neutral” thiolreducing agents are weak acids (with pK_(a)'s around pH 8-9). Thus, atbasic pH, these reducing agents migrate into the gel and, if present atsufficient concentration, provide some protection against oxidation ofsulfhydryls. At a neutral separating pH, DTT from the sample buffer isin an uncharged form and will remain behind in the sample well. Thus, noreducing agent migrates into the gel.

To maintain proteins in a reduced form during electrophoresis at neutralpH, it was found advantageous to use a reducing agent that would migrateinto the gel at neutral pH. Sodium bisulfite (2-10 mM) was found tomaintain a reducing environment in the gel during electrophoresis. Fullyreduced TGA (or similar negatively charged thiols) give similar resultsat comparable concentrations. However, partially oxidized TGA willpromote partial oxidation of protein thiols. Because reduction(oxidation) of protein thiols will take place via disulfide interchange,the ratio of reduced to oxidized thiols in the protein willsubstantially reflect the ratio of reduced to oxidized thiols in theTGA. Conversely, sulfite oxidizes to sulfate, which does not participatein redox reactions under conditions found in the gel. Therefore,regardless of the sulfite/sulfate ratio in a partially oxidizedpreparation of sulfite, as long as sufficient sulfite remains, proteinswill be protected against thiol oxidation.

It was also found that Tris could be substituted for Bis-Tris in therunning buffer with no visible effect on the separation quality.Bis-Tris may be preferred where the protein will be intentionallymodified post-separation. Bis-Tris is a tertiary amine and will notinterfere with the protein modifying agents which react through primaryamines. Tris, however, is the preferred choice for routine use, becauseit is available at significantly lower cost than Bis-Tris.

Example 7

A 14.7% T/5% C TBE urea gel was made in the following manner. To preparethe separating gel solution, a 30% acrylamide/1.6% bis-acrylamide stocksolution (47.5 ml), and a 5× gel buffer stock solution containing 0.45 MTris, 0.45 M boric acid, and 0.01 M EDTA, pH 8.18 (20 ml) were mixedwith urea (36 g), TEMED (20 ul), and enough water was added to make 100ml. The final solution pH was 8.87. It was degassed, a 10% ammoniumpersulfate solution (“10% APS”) (12.8 ul) was added to 6.4 ml, andpoured into a 1 mm thick mini-gel cassette from NOVEX (San Diego,Calif.). A comb-forming gel solution was made similarly, with thefollowing differences: acrylamide/bis solution (12.7 ml), TEMED (50 ul),and no urea; to 1.0 ml of this solution was added 10% APS (0.1 Ml) andit was immediately poured on top of the separating gel solution. A 1 mm10-well comb (NOVEX) was added, and the gels were allowed to polymerizefor at least 30 minutes at room temperature. They were then run afterstorage in sealed pouches with 1× gel buffer containing 7M at differenttemperatures.

The gels were run fresh or after storage at either 4° C. or 35° C.Samples employed were a 10b oligo DNA standard (BRL, Bethesda, Md.) oran 18-mer custom-synthesized DNA fragment (Synthetic Genetics, SanDiego, Calif.). Gels were run in an X-Cell mini-cell (NOVEX) at 180volts for 80 minutes, using 1× gel buffer in both the anode and cathodechambers. Finally, the bands were visualized by treating with Stains-Allsolution (Sigma) for 15 minutes then destaining in 20% methanol for 10minutes.

Compared to fresh gels, the gels stored at 4° C. showed a gradual lossof band sharpness and an increase in current during the electrophoresis.The loss of sharpness leads to less resolution between bands. After 2weeks at 4° C., the band width had doubled as compared to fresh gelbands. When stored at 35° C. for 1 week, the gels ran with highercurrent but the dye front only migrated 80% as far in 80 minutes. Thegel itself retained the stain, and the bands were fuzzy and indistinct.After three weeks, no bands could be seen and the gels were veryfragile.

Example 8

Gels were prepared, stored, and run as described in Example 7, exceptthat the 5× gel buffer was composed of 0.45 M Bis-Tris, 0.45 M tricine,and 0.01 M EDTA pH 7.27, the final gel solution pH was 7.70, and therunning buffer was 0.05 M Tris, 0.05 M tricine, 0.001 M EDTA. These gelsshowed no significant change in band sharpness, running current, ormigration distances when stored for up to 3 weeks at 35° C. or forseveral months at 4° C.

Example 9

Gels were prepared, stored, and run as described in Example 7, exceptthat the 5× gel buffer was composed of 0.125 M N-(2-hydroxyethyl)morpholine (HEM), 0.083 M acetic acid, and 0.002 M EDTA pH 7.0, and thefinal gel solution pH was 7.21. These gels showed no significant changein band sharpness, running current, or migration distances when storedfor up to three weeks at 35° C. or for several months at 4° C. However,the gels turned yellow on storage at 35° C.

Example 10

Mini-DNA sequencing gels were prepared from the same separating gelsolution as described in Example 7, except that only 23.3 ml ofacrylamide/bis solution was employed, the urea was increased to 42 g,and the TEMED was increased to 50 ul. It was used without degassing byadding 10% APS (50 ul) to 10 ml of the solution, and pouring between 11cm wide by 22 cm long thick glass plates with 0.25 mm spacers. The gelswere allowed to polymerize for 60 minutes at room temperature, then runthe same day.

Samples employed were an M-13 DNA sequencing reaction prepared withS³⁵-label using a USB Sequenase kit, version 2, (United StatesBiochemicals, Cleveland, Ohio). They were run in a custom-made DNAsequencing chamber at 15 watts (about 50° C.). Finally, the bands werevisualized by autoradiography. The gels had a read length of 120 baseswith a 5% error rate (95% accuracy).

Example 11

Gels were prepared and run as in Example 10, except that the 5× gelbuffer was composed of 0.5 M Bis-Tris, 0.84 M tricine, and 0.01 M EDTApH 7.2 and the final gel solution pH was 7.50. The gels had a readlength of 137 bases with a 1.5% error rate (98.5% accuracy).

Example 12

The separating gel solution was prepared as in Example 7, except that aSoaneGel SQ solution (a solution of substituted acrylamide andsubstituted bis-acrylamide cross-linkers, available from SoaneBiosciences Inc., Hayward, Calif.) was used for the polymer at 6% T, andthe TEMED was increased to 88 ul. After initiation of the gel solution(40 ml) with 10% APS (200 ul), the gels were poured in plates with 0.35mm spacers for an ABI Model 377 DNA Sequencer (Applied BiosystemsDivision of Perkin Elmer Corp., Foster City, Calif.), and allowed topolymerize at room temperature for 2 hours. The gels were loaded with apGEM sequencing reaction and run with 1× TBE buffer at 30 V/cm,generating 55° C. They had a read length of 815 bases; at 550 bases theerror rate was 1.5% (98.5% accuracy).

Example 13

Gels were prepared and run as in Example 12, except that the 5× gelbuffer was composed of 0.5 M Bis-Tris, 0.84 M tricine, and 0.01 M EDTApH 7.2, and the final gel solution pH was 7.5. The gels were run with 1×gel buffer at 30 V/cm, generating 55° C., and had a read length of 866bases; at 550 bases the error rate was 1.1% (98.9% accuracy).

Although the invention has been explained in relation to its preferredembodiments, it is to be understood that various modifications thereofwill become apparent to those skilled in the art. The foregoingdisclosure is not intended or to be construed to limit the presentinvention, or to otherwise exclude any such other embodiments,adaptions, variations and equivalent arrangements, the present inventionbeing limited only by the claims appended hereto and the equivalentsthereof.

1-22. (canceled)
 23. An electrophoresis gel comprising polyacrylamide, said gel having a shelf life of at least about one year when said gel is stored under refrigeration.
 24. The gel of claim 23, wherein said gel has a neutral pH.
 25. The gel of claim 23, wherein said gel has pH between 6 and
 8. 26. The gel of claim 25, wherein said gel is titrated to said pH with hydrochloric acid.
 27. The gel of claim 23, wherein said gel comprises from about 3% to about 25% (% T) acrylamide.
 28. The gel of claim 23, wherein said gel comprises from about 1% to about 6% (% C) cross-linker.
 29. The gel of claim 23, wherein said gel comprises an organic amine having a pK_(a) near neutrality.
 30. The gel of claim 29, wherein said organic amine is Bis(2-hydroxyethyl) iminotris (hydroxymethyl) methane.
 31. The gel of claim 23, wherein said gel is a stacking gel.
 32. The gel of claim 23, wherein said gel is a separating gel.
 33. The gel of claim 23, wherein said gel does not suffer from polyacrylamide hydrolysis during said shelf life.
 34. The gel of claim 23, wherein said gel does not suffer from decreasing resolution during said shelf life.
 35. The gel of claim 23, wherein said gel does not suffer from a change in band width of a protein electrophoresed in said gel as compared to a band width of said protein electrophoresed in a fresh gel.
 36. The gel of claim 35, wherein said band width of an electrophoresed protein changes less than 2-fold compared to said band width of said protein when said protein is electrophoresed on a fresh gel.
 37. The gel of claim 23, wherein said gel does not suffer from a decrease in migration rate of a dye front electrophoresed in said gel as compared to a migration rate of a dye front electrophoresed in a fresh gel.
 38. The gel of claim 37, wherein a dye front in said gel electrophoreses migrates at least about 80% as far as said dye front migrates in a fresh gel.
 39. The gel of claim 23, wherein said gel is capable of resolving proteins that differ in molecular weight by at least about 1,000 Daltons.
 40. The gel of claim 23, wherein said gel is capable of resolving an alkylated insulin A chain from an alkylated insulin B chain.
 41. The gel of claim 23, wherein said gel comprises at least one alkaline-labile material.
 42. The gel of claim 41, wherein said alkaline-labile material is urea or formamide.
 43. The gel of claim 23, wherein said gel is precast.
 44. The gel of claim 43, wherein said precast gel is contained within a container.
 45. The gel of claim 44, wherein said container is sealed.
 46. The gel of claim 45, wherein said container is in the shape of a cassette.
 47. The gel of claim 46, wherein said cassette further comprises a comb.
 48. The gel of claim 46, wherein said cassette is about 1 mm thick.
 49. A sealed pouch containing the precast gel of claim
 43. 50. The sealed pouch of claim 49, wherein said pouch is opaque.
 51. The sealed pouch of claim 50, wherein said pouch further contains one or more buffers.
 52. The sealed pouch of claim 51, wherein one of said one or more buffers comprises MES or MOPS.
 53. A system for electrophoresis comprising a polyacrylamide electrophoresis gel, said gel having a shelf life of at least about one year when said gel is stored under refrigeration.
 54. A method of separating biomolecules comprising electrophoresing a mixture of biomolecules through a polyacrylamide electrophoresis gel, said gel having a shelf life of at least about one year when said gel is stored under refrigeration.
 55. The method of claim 54, wherein said biomolecule is a protein, a polypeptide or a nucleic acid.
 56. The method of claim 55, wherein said protein is subject to at least one chemical modification before or during electrophoresis.
 57. The method of claim 56, wherein said chemical modification is an alkylation.
 58. The method of claim 56, wherein said chemical modification is a modification of a sulhydryl group.
 59. The method of claim 56, wherein said chemical modification is a modification of a cysteine residue.
 60. The method of claim 55, wherein said neucleic acid is selected from the group consisting of RNA and DNA.
 61. An electrophoresis gel comprising Bis(2-hydroxyethyl) iminotris (hydroxymethyl) methane and polyacrylamide, said gel having a shelf life that is greater than or equal to one month when said gel is stored under refrigeration. 